Receptor-mediated endocytosis (RME) is believed to be a major pathway for accessibility of microparticulates to their intracellular site of action. RME is controlled by polymerization of the protein clathrin at the plasma membrane to form coated vesicles, which have been implicated in cellular uptake of both liposome-encapsulated drugs and DNA for gene therapy. The goal of this proposal is to characterize the involvement of RME in microparticulate accessibility and to define further the intracellular roles of RME-related membrane transport pathways. An additional goal of this proposal is to compare microparticulate uptake in non-muscle and muscle cells, as studies of DNA uptake have suggested that differentiated muscle may have specialized uptake pathways. In concert with this last goal, we will explore the cellular function of a novel muscle-specific protein, with similarities to clathrin, that may be involved in a tissue-specific variation of RME. Previous studies funded by this center shave led us to a detailed understanding of the structure of the clathrin molecule, which we can now exploit to study its function and to study the structure and function of related molecules. The specific aims of this proposal are to 1-Express a dominant negative mutant form of clathrin in a variety of cell types to study specialized clathrin functions including liposome uptake, secretory granule formation, receptor targeting and sorting in epithelial cells; 2-Characterize the biochemical and functional properties of the novel muscle clathrin; 3-Analyze the roles of conventional clathrin and the novel muscle-specific clathrin in muscle cells with respect to endocytosis, DNA uptake and muscle differentiation. It is anticipated that these studies will reveal molecular mechanisms of microparticulate uptake that will help design strategies to increase accessibility of liposomes and DNA to the endocytic pathway. This work will also elucidate the function of clathrin and related molecules in specialized intracellular pathways in a variety of cell types.